Reduction In Open Probability Research Articles

LY294002 Inhibits Intermediate Conductance Calcium-Activated Potassium (KCa3.1) Current in Human Glioblastoma Cells.

Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion channel activity are involved to maintain the malignancy. Among them, phosphatidylinositol 3-kinase (PI3K) activity and intermediate conductance calcium-activated potassium (KCa3.1) current are involved in several aspects of GB biology. By using the electrophysiological approach and noise analysis, we observed that KCa3.1 channel activity is LY294002-sensitive and Wortmannin-resistant in accordance with the involvement of PI3K class IIβ (PI3KC2β). This modulation was observed also during the endogenous activation of KCa3.1 current with histamine. The principal action of PI3KC2β regulation was the reduction of open probability in intracellular free calcium saturating concentration. An explanation based on the “three-gate” model of the KCa3.1 channel by PI3KC2β was proposed. Based on the roles of KCa3.1 and PI3KC2β in GB biology, a therapeutic implication was suggested to prevent chemo- and radioresistance mechanisms.

Cytosolic GABA inhibits anion transport by wheat ALMT1.

Anion transport by aluminium-activated malate transporter (ALMT) proteins is negatively regulated by gamma-aminobutyric acid (GABA), which increases in concentration during stress. Here, the interaction between GABA and wheat (Triticum aestivum, Ta) TaALMT1 heterologously-expressed in Xenopus laevis oocytes was investigated. GABA inhibited anion transport by TaALMT1 in membrane patches from the cytosolic, not extracellular membrane face, via a reduction in open probability (NPopen ), not an inhibition of channel current magnitude. TaALMT1 currents in patches frequently exhibited rundown with complete removal of cytosolic factors, but were partially sustained by protein kinase C dependent phosphorylation. When applied to whole oocytes a GABA-analogue-BODIPY conjugate inhibited TaALMT1 anion currents from the cytoplasmic face only, whereas free GABA inhibited from both the inside and outside consistent with GABA traversing the TaALMT1 pore then acting from the inside. We propose GABA does not competitively inhibit ALMT conductance through the same pore but rather leads to an allosteric effect, reducing anion channel opening frequency. Across plants GABA is a conserved regulator of anion transport via ALMTs - a family with numerous physiological roles beyond Al3+ tolerance. Our data suggests that a GABA-ALMT interaction from the cytosolic face has the potential to form part of a novel plant signalling pathway.

Dialysis of an Unknown Cytosolic Cofactor Results in Partial, Delayed Activation of TRPA1

In many organ systems, a subset of sensory nerve fibers termed nociceptors are stimulated by noxious stimuli and evoke defensive sensations, behaviors and reflexes. Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in sensory neurons and evokes action potentials in nociceptive fibers when activated. Agonists of TRPA1 are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1 subunits, which result in activation. Electrophiles can modify cysteines through reversible or irreversible reactions based on the structure of the compound. Whole‐cell patch clamp and live‐cell Ca2+ imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of electrophiles. Ca2+ imaging experiments show that irreversible electrophiles like N‐ethylmaleimide (NEM) rapidly increase Ca2+ influx, which suggests TRPA1 activation. However, whole‐cell patch clamp studies show a delayed, partial activation of TRPA1 with NEM treatment, but display rapid activation by reversible electrophiles. Furthermore, inside‐out single channel patch clamp experiments exhibit a reduction in open probability with NEM treatment that is comparable to the whole‐cell patch clamp experiments. To determine the mechanism of TRPA1 activation, the cause for the disparity of TRPA1 activation between whole‐cell patch clamp and Ca2+ imaging assays must be identified. We hypothesized that dialysis of cellular components in whole‐cell patch clamp that promote rapid, robust TRPA1 activation with NEM. To test our hypothesis, I performed whole‐cell patch clamp and gramicidin‐perforated patch clamp, a non‐invasive patch clamp method, to observe and compare the rate‐of‐change of NEM‐induced TRPA1 responses. Both methods contain 5mM Na‐polyphosphates in the pipette solution to prevent rundown of the TRPA1 channel. Studies using gramicidin‐perforated patch clamp exhibit a significant increase in the rate‐of‐change of NEM‐induced TRPA1 responses compared to activation in whole‐cell experiments. We also hypothesize that glutathione is the unidentified cofactor that is dialyzed out of the cell in whole‐cell experiments. Future whole‐cell experiments with glutathione (5mM) in the pipette solution will be performed to determine if rapid TRPA1 activation can be rescued. Identification of the unknown cytosolic cofactor will promote a complete understanding of the electrophilic cysteine modification events that underlie TRPA1 activation.Support or Funding InformationR01 from the NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calmodulin Regulation of Ryanodine Receptors (RyR2) Differs in Failing and Non-Faling Human Hearts due to Differences in RyR2 Phosphorylation

The RyR2 is a macromolecular complex comprising a Ca2+ channel and many accessory proteins that regulate channel activity. In animal models, the binding of intracellular calmodulin (CaM, ∼45 nM in the cytoplasm) partially inhibits calcium release in cardiomyocytes due to its ability to inhibit RyR2 channel opening. Single channel studies of RyR2 in lipid bilayers find that CaM inhibits RyR2 from animal hearts with an IC50 of 100 nM.In this study, RyR2 was isolated from healthy human donor hearts and hearts with ischemic cardiomyopathy (ICM) with ethics approval (UoN H-2009-0369; Univ Sydney #2012/2814; QUT EC28114) and incorporated into lipid bilayers. We found that RyR2 from healthy human hearts were not inhibited by CaM at concentrations up to 500 nM whereas for RyR2 from human hearts with ICM, CaM caused a reduction in open probability of 57% ± 6%. Dephosphorylating these RyR2 channels with Protein Phosphatase 1 prior to experiments abolished the effect of CaM, suggesting that increased RyR2 phosphorylation in ICM hearts mediates CaM inhibition in humans.

Mg2+ modulation of the single-channel properties of KCa3.1 in human erythroleukemia cells

The activity of many ion channels is modulated by ions other than the ones they primarily conduct, with important consequences for cell signalling. In this study, we demonstrate that Mg(2+) inhibits the intermediate conductance calcium-activated potassium channel (KCa3.1) in human erythroleukemia cells via two distinct mechanisms. Firstly, intracellular Mg(2+) blocks this channel via a rapid, voltage-dependent mechanism that leads to a reduction of the channel's unitary current. We show that this block involves interactions which are well described by the Woodhull model. Secondly, we found that Mg(2+) reduces the open probability of the channel. By analysing the channel kinetics, we found that this reduction in open probability is at least partly due to a reduction in the rate of channel opening from the closed state, a finding that can be accounted for if Mg(2+) competes with Ca(2+) for the activation site. Consistent with this interpretation, we find that the decline in relative NPo observed in the presence of 5mMMg(2+) could be significantly reduced by increasing the free Ca(2+) concentration.

Neural Cell Adhesion Molecule-associated Polysialic Acid Inhibits NR2B-containing N-Methyl-d-aspartate Receptors and Prevents Glutamate-induced Cell Death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.

Functional NR2B‐ and NR2D‐containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 nm) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 nm or 200 microm NMDA was not affected by zinc or TPEN (N,N,N',N'-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 microm ifenprodil reduced open probability, while 10 microm reduced channel open time but had no effect on open probability of channels activated by 100 nm NMDA. When the concentration of NMDA was increased to 200 microm, ifenprodil (10 microm) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.

Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line.

1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.

Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: chloride imaging and cell-attached patch-clamp recordings.

The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl-. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (-)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (tau0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (tau(c1) = 0.7 ms, tau(c2) = 25.4 ms) consistent with at least two closed states. Using a Cl- -sensitive dye to measure intracellular [Cl-], it was found that perfusion with gluconate-containing, low Cl- medium depleted intracellular [Cl-]. It was therefore possible to reduce intracellular [Cl-] by perfusion with a low Cl- solution while maintaining the extracellular channel surface in high Cl- pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl- solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl-] may thus modulate photoreceptor Ca2+ channels.

Bombesin Receptors Inhibit G Protein-Coupled Inwardly Rectifying K+Channels Expressed inXenopusOocytes through a Protein Kinase C-Dependent Pathway

Although activation of G protein-coupled inward rectifying K+ (GIRK) channels by Gi/Go-coupled receptors has been shown to be important in postsynaptic inhibition in the central nervous system, there is also evidence to suggest that inhibition of GIRK channels by Gq-coupled receptors is involved in postsynaptic excitation. In the present study we addressed whether the Gq-coupled receptors of the bombesin family can couple to GIRK channels and examined the mechanism by which this process occurs. Different combinations of GIRK channel subunits (Kir3.1, Kir3.2, and Kir3.4) and bombesin receptors (BB1 and BB2) were expressed in Xenopus oocytes. In all combinations tested GIRK currents were reversibly inhibited upon application of the bombesin-related peptides, neuromedin B or gastrin-releasing peptide in a concentration-dependent manner. Incubation of oocytes in the phospholipase C inhibitor U73122 or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine significantly reduced the inhibition of GIRK currents by neuromedin B, whereas the Ca2+ chelator, BAPTA-AM had no effect. The involvement of PKC was further demonstrated by direct inhibition of GIRK currents by the phorbol esters, phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate. In contrast, the inactive phorbol ester 4alpha-phorbol and protein kinase A activators, forskolin and 8-bromo cAMP did not inhibit GIRK currents. At the single-channel level, direct activation of PKC using phorbol ester phorbol-12, 13-dibutyrate caused a dramatic reduction in open probability of GIRK channels due to an increase in duration of the interburst interval.

Interaction between the pore and a fast gate of the cardiac sodium channel.

Permeant ions affect a fast gating process observed in human cardiac sodium channels (Townsend, C., H.A. Hartmann, and R. Horn. 1997. J. Gen. Physiol. 110:11-21). Removal of extracellular permeant ions causes a reduction of open probability at positive membrane potentials. These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel. We tested this hypothesis by three sets of manipulations designed to affect the binding of cations within the pore: application of intracellular pore blockers, mutagenesis of residues known to contribute to permeation, and chemical modification of a native cysteine residue (C373) near the extracellular mouth of the pore. The coupling between extracellular permeant ions and this fast gating process is abolished both by pore blockers and by a mutation that severely affects selectivity. A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations. Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.

Properties and functions of calcium-activated K+ channels in small neurones of rat dorsal root ganglion studied in a thin slice preparation.

1. Properties, kinetics and functions of large conductance calcium-activated K+ channels (BKCa) were investigated by the patch-clamp technique in small neurones (Adelta- and C-type) of a dorsal root ganglion (DRG) thin slice preparation without enzymatic treatment. 2. Unitary conductance of BKCa channels measured in symmetrical high K+ solutions (155 mM) was 200 pS for inward currents, and chord conductance in control solution was 72 pS. Potentials of half-maximum activation (V ) of the channels were linearly shifted by 43 mV per log10 [Ca2+]i unit (pCa) in the range of -28 mV (pCa 4) to +100 mV (pCa 7). Open probabilities increased e-times per 15-32 mV depolarization of potential. 3. In mean open probability, fast changes with time were mainly observed at pCa > 6 and at potentials > +20 mV, without obvious changes in the experimental conditions. 4. BKCa channels were half-maximally blocked by 0.4 mM TEA, measured by apparent amplitude reductions. They were completely blocked by 100 nM charybdotoxin and 50 nM iberiotoxin by reduction of open probability. 5. Two subtypes of small DRG neurones could be distinguished by the presence (type I) or absence (type II) of BKCa channels. In addition, less than 10 % of small neurones showed fast (approximately 135 V s-1) and short ( approximately 0.8 ms) action potentials (AP). 6. The main functions of BKCa channels were found to be shortening of AP duration, increasing of the speed of repolarization and contribution to the fast after-hyperpolarization. As a consequence, BKCa channels may reduce the amount of calcium entering a neurone during an AP. 7. BKCa channel currents suppressed a subsequent AP and prolonged the refractory period, which might lead to a reduced repetitive activity. We suggest that the BKCa current is a possible mechanism of the reported conduction failure during repetitive stimulation in DRG neurones.

A mutation in the pore region of HERG K+ channels expressed in Xenopus oocytes reduces rectification by shifting the voltage dependence of inactivation.

1. The effects of a mutation in the human ether-a-go-go-related gene (HERG) (Ser631 to Ala, S631A) on the voltage- and extracellular [K+] dependence of inactivation were studied in Xenopus oocytes using two microelectrode and single channel voltage-clamp techniques. 2. The voltage required for half-inactivation of S631A HERG was 102 mV more positive than for wild-type (WT)-HERG, resulting in reduced rectification of the steady-state current-voltage relationship. In contrast, the voltage dependence of channel activation was not altered by the S631A mutation. These findings indicate that inactivation of HERG channels is not linked to activation. 3. Rectification of whole-cell S631A HERG current was caused by a voltage-dependent reduction in open probability, and inward rectification of the current-voltage relationship of single channels. 4. Elevation of extracellular [K+] from 2 to 20 mM shifted the half-point for inactivation by +20 mV for WT-HERG, and +25 mV for S631A HERG. Thus, elevated [K+]o and the S631A mutation affect HERG inactivation by different mechanisms. 5. The S631A mutation altered the ion translocation rate of HERG channels. The single channel conductance (gamma) of S631A HERG was 20 pS between -40 and-100 mV, and 6.0 pS between +40 and +100 mV (120 mM extracellular K+). This compares to a gamma of 12.1 and 5.1 pS for WT-HERG channels under the same conditions.

Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability

The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations.

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4-10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.

Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+ and Na+ channels.

The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryloxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-insensitive Na+ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca2+ or Na+ channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca(2+)-channel specific: DTDP induced no changes in expressed human cardiac Na+ current. Single-channel Ba2+ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca2+ current reflects changes in gating and not permeation. In summary, the pore-forming (alpha 1) subunit of the L-type Ca2+ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

Effects of disopyramide and flecainide on the kinetics of inward rectifier potassium channels in rabbit heart muscle.

1. Standard patch-clamp techniques were used to study the interaction of therapeutic concentrations of flecainide and disopyramide with single inwardly-rectifying potassium channels in cell-attached membrane patches from rabbit ventricular myocytes. 2. Under drug-free conditions, the potassium channels had a conductance of 31 +/- 2 pS (n = 13), a mean open time of 230 +/- 6 ms (n = 11) recorded at the resting cell potential, and an open probability of 0.66 +/- 0.20 (n = 39). The resting potential of the cells studied was -68.5 +/- 3.6 mV (n = 32). 3. Disopyramide did not reduce the open probability of the channel when the cell was voltage-clamped at the resting cell potential. However, disopyramide increased the mean open time of the channel, recorded at the resting cell potential, by 15% at 5 microM and by 29% at 20 microM. The action potential prolonging actions of disopyramide in therapeutic concentrations appear not to be due to blocking the inward rectifier K+ channel. 4. Flecainide (3.0 microM, but not at 0.5 microM) decreased the open probability without changing the conductance of the channel, at 3 microM (51.0 +/- 7.2%, n = 6, P = 0.03) at the resting cell potential. Flecainide increased the mean open time of the channel, recorded at the resting cell potential, by 12% at 3.0 microM. 5. We propose that flecainide stabilized the inward rectifier K+ channel in an inactivated state, without plugging the conducting pore. In addition, it appeared to bind to an open conformation of the channel,since some of the reduction in open probability could be accounted for by the lengthening of the mean open time. The changes in open-state kinetics suggest that this binding may be in the region of the activation gate.

Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons

Recent data have indicated that the divalent cation Zn2+ can selectively block central neuronal excitation mediated by N-methyl-D-aspartate (NMDA) receptors. The present experiments were conducted to determine the action of Zn2+ at the single-channel level. Outside-out membrane patches were prepared from cultured murine cortical neurons. Glutamate, 3 microM, in the presence of 5 microM glycine activated channels with a main conductance state of about 50 pS which were blocked in a voltage-dependent manner by Mg2+. Zn2+ appeared to have 2 effects on these NMDA receptor-activated channels. First, at concentrations as low as 1-10 microM, Zn2+ produced a concentration-dependent reduction in channel open probability, insensitive to membrane voltage between -60 and +40 mV; about 50% reduction in open probability was produced by 3 microM Zn2+. This reduction was mostly due to a decrease in opening frequency and only weakly mimicked by Mg2+. Second, at higher concentrations (10-100 microM) and negative membrane voltages, Zn2+ additionally produced an apparent reduction in single-channel amplitude, associated with an increase in channel noise, suggestive of a fast channel block. The amplitude reduction was voltage-dependent, with a delta of 0.51; amplitude distribution analysis suggested that this voltage dependence was primarily contributed by the "on" blocking rate constant, with little contribution from the "off" rate constant. The channel block produced by Zn2+ was faster than that of Mg2+, which at 100 microM and negative membrane voltages induces flickering of the NMDA receptor-activated channel without changing apparent channel amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)